Nearly two-thirds of the body's iron endowment is in the form of hemoglobin and each red blood cell contains more than a billion iron atoms in the form of heme. Consequently, it is not surprising that inherited or acquired defects in hemoglobin synthesis, including the thalassemias, hemoglobinopathies, iron deficiency, and the anemia of inflammation are among the most prevalent human afflictions. Importantly, most of the ~25 mg of iron required to synthesize the hemoglobin in ~360 billion RBCs each day (5 million/sec) derives from recycling of heme-iron from aged RBCs catabolized by reticuloendothelial system (RES) macrophages; at steady state, dietary heme and non-heme iron absorption contribute only 1-2 mg/day to the systemic iron economy, and up to two-thirds of this is absorbed as heme-iron. Nevertheless, despite the importance of RES iron recycling and intestinal heme absorption, the molecular pathways of transmembrane heme transport in macrophages and intestinal epithelial cells have, for the most part, remained unknown. This is in large part due to technical difficulties in identifying heme-specific transporters in mammalian cells and the inability to translate these finding to whole organisms. Recently, using C. elegans, which bypasses several of the technical hurdles presented by mammalian cells, our collaborators identified a protein, heme-responsive gene 1 (hrg-1), which functions a heme transporter. Our preliminary in vitro and in vivo data strongly suggest that the mammalian ortholog of hrg-1, HRG1, is involved in macrophage iron recycling, intestinal heme absorption, as well as intraerythroid and hepatocellular heme metabolism. Here, we propose to investigate the in vivo function of Hrg1 in the mouse by examining its regulation in macrophages and intestinal epithelial cells in response to heme and iron, as phagocytosed red blood cells, as well inflammation, all of which modulate systemic iron metabolism and contribute to the pathogenesis of common nutritional, acquired, and inherited anemias. In addition, we will directly test the hypothesis that Hrg1 is essential for macrophage iron recycling, intestinal heme absorption, and intraerythroid iron metabolism using conditional gene targeting technology in the mouse. We expect that the collective results will yield substantial insight into these hitherto obscure aspects of heme and iron biology that are critical to our understanding and rational treatment of these common diseases.